Sepsis is characterized by systemic activation of coagulation and inflammation in response to microbial infection. Although cell-free DNA (cfDNA) released from activated neutrophils has antimicrobial properties, it may also exert harmful effects by activating coagulation and inflammation. The authors aimed to determine whether deoxyribonuclease (DNase) administration reduces cfDNA levels, attenuates coagulation and inflammation, suppresses organ damage, and improves outcome in a cecal ligation and puncture (CLP) model of polymicrobial sepsis. Healthy C57Bl/6 mice were subjected to CLP, a surgical procedure involving two punctures of the ligated cecum, or sham surgery (no ligation/puncture). Mice were given DNase or saline by intraperitoneal injection 2, 4, or 6 h after surgery. Two hours after treatment, organs were harvested and plasma levels of cfDNA, interleukin-6 (IL-6), IL-10, thrombin-antithrombin complexes, lung myeloperoxidase, creatinine, alanine transaminase, and bacterial load were quantified. Survival studies were also performed. The CLP-operated mice had rapid time-dependent elevations in cfDNA that correlated with elevations in IL-6, IL-10, and thrombin-antithrombin complexes and had organ damage in the lungs and kidneys. Administration of DNase at 2 h after CLP resulted in increased IL-6 and IL-10 levels and organ damage in the lungs and kidneys. In contrast, DNase administration at 4 or 6 h after CLP resulted in reduced cfDNA and IL-6 levels, increased IL-10, and suppressed organ damage and bacterial dissemination. Deoxyribonuclease administration every 6 h after CLP also rescued mice from death. Our studies are the first to demonstrate that delayed but not early administration of DNase may be protective in experimental sepsis.

BackgroundDespite increasing ethical standards for conducting animal research, death is still often used as an endpoint in mouse sepsis studies. Recently, the Murine Sepsis Score (MSS), Mouse Clinical Assessment Score for Sepsis (M-CASS), and Mouse Grimace Scale (MGS) were developed as surrogate endpoint scoring systems for assessing pain and disease severity in mice. The objective of our study was to compare the effectiveness of these scoring systems and monitoring of body temperature for predicting disease progression and death in the cecal ligation and puncture (CLP) sepsis model, in order to better inform selection of surrogate endpoints for death in experimental sepsis.MethodsC57Bl/6J mice were subjected to control sham surgery, or moderate or severe CLP sepsis. All mice were monitored every 4 h for surrogate markers of death using modified versions of the MSS, M-CASS, and MGS scoring systems until 24 h post-operatively, or until endpoint (inability to ambulate) and consequent euthanasia.ResultsThirty percent of mice subjected to moderate severity CLP reached endpoint by 24 h post-CLP, whereas 100% undergoing severe CLP reached endpoint within 20 h. Modified MSS, M-CASS, and MGS scores all increased, while body temperature decreased, in a time-dependent and sepsis severity-dependent manner, although modified M-CASS scores showed substantial variability. Receiver operating characteristic curves demonstrate that the last recorded body temperature (AUC = 0.88; 95% CI 0.77–0.99), change in body temperature (AUC = 0.89; 95% CI 0.78–0.99), modified M-CASS (AUC = 0.93; 95% CI 0.85–1.00), and modified MSS (AUC = 0.95; 95% CI 0.88–1.01) scores are all robust for predicting death in CLP sepsis, whereas modified MGS (AUC = 0.78; 95% CI 0.63–0.92) is less robust.ConclusionsThe modified MSS and body temperature are effective markers for assessing disease severity and predicting death in the CLP model, and should thus be considered as valid surrogate markers to replace death as an endpoint in mouse CLP sepsis studies.